In a general manner, the invention relates to a sequence of DNA performing a function which is expressed in an over-production of extracellular proteins by various strains of Bacillus, as well as vectors containing this sequence.
The invention relates more especially to a sequence of DNA coding for a polypeptide acting on the over-production of extracellular enzymes, in particular of alkaline protease, by B. subtilis.
In fact, the growing interest directed in recent years toward the use of B. subtilis as a host for cloning, and destined in particular for the production of extracellular proteins, can be explained on the following grounds. Firstly, the genetic characterization of B. subtilis has been carried out to a large extent, and effective methods for the introduction and extraction of DNA fragments as well as selective markers of resistance to certain antibiotics have already been described. Moreover, owing to its properties of sporulation, B. subtilis represents a good model of procaryotic differentiation.
From a practical point of view, one of the interesting properties of this organism is that it can be handled in complete safety because it does not infect humans or animals.
Moreover, it possesses the property of secreting a large number of enzymes (Bacteriological Reviews: 41, 711-753 (1977), some of which, such as .alpha.-amylase, proteases and glucanases, are particularly interesting at the industrial level. In particular, the alkaline protease is very much used in the manufacture of detergents as well as in the pharmaceutical industry and the agri-foodstuffs industry.
The techniques for the recombination of DNA have also made it possible to use B. subtilis as a host which secretes foreign proteins. In these constructions, the gene corresponding to the protein in question is fused with a sequence which allows its expression and its secretion by B. subtilis, the said sequence being also referred to by the expression "signal sequence" coding for a signal peptide. Numerous examples have been described which use the regulatory sequences as well as the regions corresponding to the signal peptides of .alpha.-amylase, alkaline protease and the neutral protease of B. subtilis (J. Bacteriol., 165, 837-842, (1986)), B. amyloliquefaciens and B. licheniformis.
However, although B. subtilis seems to be a receptor host of choice as a result of its being well characterized, the levels of secretion for this organism are much lower if they are compared with those of Bacillus strains used in industry such as B. licheniformis, B. amyloliquefaciens or B. stearothermophilus which are capable of producing large amounts of secreted enzymes.
A typical example is that of alkaline protease.
The B. subtilis 168 strain placed in culture under laboratory conditions and in an optimal culture medium can produce 50 to 100 units of azocasein per ml of supernatant of an activity of the type of that of alkaline protease.
The industrial strain B. licheniformis (deposited in the National Collection of Cultures of Micro-organisms at the Pasteur Institute in Paris under the number 71 I 001) grown in that same optimal culture medium can produce more than 100,000 units of azocasein per ml, thus in a yield multiplied by a factor of 1,000.
It is possible that such strains have accumulated mutations which alter the properties of the secreted enzyme such as thermostability, and/or mutations which increase the levels of secretion and synthesis of proteins.
During recent years, detailed studies have been carried out in order to increase the production of alkaline protease in the well characterized strain of B. subtilis 168. In this way, a large number of mutants have been isolated which increase the secreted levels of proteolytic enzymes.
In the first instance, hpr mutants were characterized by their over-production of alkaline and neutral proteases (J. Bacteriol.: 112, 1026-1028 (1972)).
Sac U.sup.h mutants (Biochimie; 56, 1481-1489, (1974)), identical with pap mutants (J. Bacteriol.: 161, 1182-1187 (1985), and J. Bacteriol.: 124, 48-54, (1975)), have also been described as possessing a pleiotropic phenotype for the over-production of levansucrase, protease and .alpha.-amylase.
Furthermore, the regulatory locus nprR2 has been described as being responsible for the specific stimulation of the secretion of the neutral protease (i. Bacteriol.: 139, 583-590, (1979), and J. Bacteriol.: 119, 82-91, (1974)). However, the molecular bases of these mutations still remain unknown since it has not been possible to clone any of the mutated allelic forms of the genes implicated in the observed phenotype. That is the reason why it is very difficult to transfer these mutations to other species of Bacillus so as to increase their productive capacity.
Several research teams have recently described the cloning of DNA fragments which make possible the hypersecretion of protease in B. subtilis.
The Japanese patent filed on Jul. 29, 1983 by Okada et al. under the No. 60-30 685 describes a recombinant DNA containing an inserted sequence derived from the genome of a B. licheniformis presented as coding for a protease of this latter type of micro-organism. The authors of the patent have used this recombinant DNA to transform cultures of Bacillus subtilis with a view to increasing their capacity to produce alkaline and neutral proteases.
Okada et al. (Appl. Microbiol. Biotechnol.: 20, 406-412, (1984)) have cloned a EcoRI fragment of 3.6 kb from B. licheniformis which stimulates the secretion of both the neutral protease and the alkaline protease (70.times.), but which has only a slight effect on the enzymes of the .alpha.-amylase type (2.times.); the observed phenotype is conserved when a pvuII fragment of 1.25 kb and even a Sau 3A1 fragment of 0.37 kb obtained from the EcoRI fragment are cloned. The authors do not exclude the possibility that the cloned sequence belongs to a regulator gene rather than to genes coding for proteases.
Tomioka et al. (J. Biotechnol.: 3, 85-96, (1985)) have also described the stimulation of the secretion of neutral (10.times.) and alkaline (9.times.) proteases with a EcoRI fragment of 4 kb derived from B. amyloliquefaciens; it was possible to reduce this fragment to 1.75 kb containing non-identified, open-reading frames; it did not lead to stimulation of the secretion of .alpha.-amylase.
A fragment of DNA derived from Bacillus natto has been described as leading to an over-production of neutral and alkaline proteases and of levansucrase (J. Bacteriol.: 166, 20-29, (1986)). The authors thought it likely that a polypeptide of 60 amino acids could be responsible for the stimulation of this over-production.
Finally, the identification of the sacQ gene, a pleiotropic gene affecting the expression of a large number of genes coding for products secreted by various strains of Bacillus as well as the identification of polypeptides of 46 amino acids encoded in this sacQ gene have recently been described in B. subtilis (J. Bacteriol.: 166, 113-119, (1986)), and in B. amyloliquefaciens (J. Biotechnol.: 3, 85-96, (1985)).